Novel glucuronic acid derivatives and process for the preparation thereof

ABSTRACT

FURAN   2-(R1-O-),3,4-DI(HO-),5-(R2-NH-NH-CO-CH(-OH)-)TETRAHYDRO-   NOVEL GLUCURONIC ACID DERIVATIVES OF THE FORMULA WHEREIN R1 AND R2 ARE AS DEFINED ABOVE.   H2N$NH-R2   WITH A PHENYLHYDRAZINE COMPOUND OF THE FORMULA   2-(R1-O-),3-HO,4,5-(-O-CO-CH(-OH)-)TETRAHYDROFURAN   WHEREIN R1 IS LOWER ALKYL GROUP, AND R2 IS A PHENYL GROUP WHICH MAY CONTAIN ONE ORE MORE SUBSTITUENTS SELECTED FROM THE GROUP CONSISTING OF A LOWER ALKYL GROUP, A LOWER ALKOXY GROUP, A NITRO GROUP ANDA HALOGEN ATOM, WHICH ARE USEFUL AS A PHARMACEUTICAL, IN PARTICULAR, FOR THE PREVENTION AND TREATMENT OF LIVER FUNCTION DISORDERS, ARE DISCLOSED. THE GLUCURONIC ACID DERIVATIVES CAN BE PREPARED BY REACTING AN ALKYL-D-GLUOFURANOSIDURONOACTONE OF THE FORMULA

NOV. 27, 1973 os ()KUI EI'AL 3,775,396 NOVEL GLUCURONIC ACID DERIVATIVES AND PROCESS FOR THE PREPARATION THEREOF Filed May 12, 1971 4000 3000 20'00l8'00 \e'00|4'00 IOOIObOBbOGU) cm-l { M/H 6; ,INVENTOR ORNEYS United States Patent (Mike NOVEL GLUCURONIC ACID DERIVATIVES AND US. Cl. 260-210 28 Claims ABSTRACT OF 'rnnmscLosunn Novel glucuronic acid derivatives of the formula CO-NHNH-Rz wherein R is a lower alkyl group, and R, is a phenyl group which may contain one ormore substituents selected from the group consisting of a lower alkyl group, a lower alkoxy group, a'nitro group and a halogen atom,

' which are useful as a pharmaceutical, in particular, for

the prevention and treatment of'liver function dlsorders,

are disclosed. The glucuronic acid derivatives can be prepared by reacting an alkyl-D-glucofuranosiduronolactone of the formula Col...

. H-ORr l t l l. with a phenylhydrazine compound of the formula H N-NH R wherein R and R are as defined above.

This invention relates to novel glucuronic acid derivatives' of the formula g V OO-NHNH-Rr o l l H-OR: (in

wherein R is a lower alkyl group, and R is a phenyl group which may contain one or more substituents selected from the group consisting of a lower alkyl group, a

lower alkoxy group, a nitro group and a halogen atom, and to a process for the preparation thereof.

In "accordance with the present invention," the" novel glucuronic acid derivatives represented by the above formula are prepared by reacting an allgyl-Dglucofuranosiduronolactone of the formula 3,775,396 Patented Nov. 27, 1973 wherein R is as defined above, when a phenylhydrazine compound of the formula H N'NHR wherein R is as defined above.

The btarting material, alkyl-D-glucofuranosiduronolactone, used in the process of this invention can be prepared by the conventional procedures. For example, it can easily be prepared by reacting D-glucuronolactone with a selected alcohol in the presence of an acidic ion-exchange resin or hydrogen chloride as a catalyst.

The process of this invention can be carried out by heating the reactants alkyl-D-glucofuranosiduronolactone and phenylhydrazine compound. If necessary, the reactants may be dissolved in an appropriate organic solvent such as alcohol, chloroform or the like. The phenylhydrazine compound is preferably used in an equimolar amount or in excess with respect to the alkyl-D-gluco furanosiduronolactone. The temperature and the period required for completing the reaction depend upon the type of the reactants, solvent used, etc., but are usually approximately in the range of from one-half to six hours at or near the refluxing temperature of the reaction mixture.

The starting material, alkyl-D-glucofuranosidurono1ac- I tone may be used in an isolated and purified form or, alternatively, may be a reaction mixture of the abovedescribed reaction of D-glucuronolactone and an alcohol, from which the catalyst has been removed. In the latter case, the process of this invention can be carried out by adding a phenylhydrazine compound to the reaction mixture followed by heating.

The thus obtained alkyl-D-glucuronosidephenylhydrazides of this invention, which are noval compounds not previously disclosed in literature, exhibit low toxicity and high stability and are extremely useful as pharmaceuticals, in particular, for the treatment of liver function disorders.

It is to be understood that the product and the starting alkyl-D-glucofuranosiduronolactone of this invention may exist in aor B-form. The present invention includes within its scope the mixture of such aand fi-forms as well as a separated uor p-form.

FIG. 1 is an infrared absorption spectrum of ethyl-D- glucuronoside-p-ethoxyphenylhydrazide in a-form.

FIG. 2 is an infrared absorption spectrum of ethyl-D- glucuronoside-p-ethoxyphenylhydrazide in ,B-form.

The present invention is further illustrated by the following experiment and examples but these are given for exemplifying the specific embodiments only and are not to be construed as limiting the scope of the invention.

EXPERIMENT 1 The pharmacological activity of the compounds of this invention are shown in Table 1. The percent prevention of hepato-cellular necrosis was determined as follows:

" Mice were administered test compounds (per os) together with 0.2 ml./mg. (body weight) ofcarbontetrachloride (i.p.)' anaafierzb hours of such administrations, Evans Blue dye-was administered intravenously. After 30 minutes of thedye administration, liver wasexcised from the sacrificed mice and the amount of dye which had penetrated into the liver was determined. Two control groups were also used, one receiving only carbon tetrachloride and other receiving neither test compound nor CCl The percent prevention of hepato-cellular necrosis (average of five mice in each case) was calculated by the following formula: Percent-prevention hepato cellu'la'r necrosis A=dye amount in the group receiving test compound+CCl B=dye amount in the untreated control group.

C=dye amount in the group receiving only CCl After 24 hours, serum transaminase (glutamic-oxalacetic transaminase and glutamic-pynuvic transaminase) activities were determined. The results are shown in the following table.

TABLE 1 TABLE 3 Prevention No. Mean=l=S.D.

of he atof Dose ce ular rats GOT l GPT 1 (mole! necrosis Compound kg.) (percent) GllillgA pEPH plus C014 (0.5 ml./ 4 13 1'0 15 1 :1: :l: Methyl-B-D-glucuronoside-p-to1ylhydrazide-. 0.5 100 coil 0.5 mLIkg. only 4 226:1:83 5a+12i 0. 25 s0 and. p-EPH plus 001. (l 5 ml/ 0.20 50 kg.)

4 55:1:24 (2) 12:1:3} (2) 0.1 0 0014, 1.5 ml./k nly 4 167=|=75 55:1:40

Normal control 6 20:6 Hi4 Ethyl-fi-D-glucur0noside-p-chlorophenylhydrazide 0. 5 100 l Ker-men units in 50 times diluted serum.

0.01 4 As is clear from the table, the elevation of serum trans- Eth l-D-glucuronoside-p-tolylhydrazlde.1/2 amlnase activities was reversed to normal by the admindizlmnfi e 0. 25 100 lstr-atlon of GFUA p-EPH.

58 (c) Bromosulfophthalein (herein called as BSP) reten- 0. 05 0 tlon (mouse): Five or six male mice (ddY-strain, -30 Ethyl-D-glucuronosidephenylhydre.zide...-: 0.5 100 hiwmg recewed A F mgjkgfi) 0.25 so minutes before, were in ected with CC1 lntraperltoneally i After 24 hours, BSP (50 mg./kg.) was in ected mtran'Butyl'n'glummmsidephenymydm1m-=- 85% 25 venously to the mice and plasma BSP concentration was 0. 0s 30 determined at 10 minutes after BSP injection with optical 0 densities at 570 me of the alkalim'zed plasma. The results n-Butyl-D-glucuronoside-p-tolylhydrazide..:: 0 8.71) 12 are shown in the following table.

0.05 0 30 TABLE 4 t l-D- lu id 111 h l- Number BSP, mean salons..ft feiiiaifffiria...... on... ow... 0102s 30 GFUA p-EPH, 0.2 m1. Coll/ g 7 6 0 027:1:0. 010

0.01 0 0.21111. coll/kg. only 5 0 224i0.085 GFUA -EPH, 0.51111. CC1l/kg.-- e 0 025:1:0010 Ethyl-a-D-glueuronoslde-p-ethoxyphenyl- 0.5 ml- Clllkg. only 6 0 293:1:0. 093 hydrazlde 0 18% Normal control 5 0 020:1:0. 007 828% 23 (d) BSP retention (rat): Three, five or six male rats 0.01 25 (Wistar-Imamichi strain, 200-250 g.), having received GFUA p-EPH (100 mg./kg.) orally 1 hour before were --D-l idth h 1- l ilzzid it f?f fi ui. f..?i?.: 3Y.--.;; 0.25 100 40 in ected wlth CCl (1.0 mL/kg.) mtraperltoneally.

95 After 24 hours, BSP (100 mg./kg. was injected intra- 0. 05 9s 0.03 90 veneously to the mice and plasma BSP concentration 9 f8 was determined at minutes after BSP injection. The

results are shown in the following table. 45 EXPERIMENT 2 TABLE 5 The effect of ethyl-D-glucuronoside-p-etholtypherlylhy- Number i as nl a drazide (a mlxture of 0tand fl-forms, herein called as of rats percent P' against Vafious exWmmmtal 5%;. ngg, g mg.I/]lg., p.o lg g.

- m .o rtniunes 14 d l S gg un g-nrn, 1o0ni 1 .o g 3 55.5 1) CC -induce acute iver injuries.- a erum' 40 y transammase (mouse): Five male rmce (ddY-straln, Normalwnml 3 4 25-30 g.), having received GFUA p-EPH (35.6 mg./kg.) orally 30 minutes before, were injected with CCl; (0.2 (e) Fatty liver (mouse): Male mice (ddY-strain, mg./kg.) intraperitoneally. 20-26 g.), starved for 17 hours before the experiment,

After 22 hours, serum transaminase (glutamic-oxalwere used. acetic transaminase and glutalnic-pyruvic transaminase) Four, five or six mice, havlngrecelved GFUA p-EPH activities were determined. The results are shown in the (50 mg./kg.) orally 30 minutes before, were lnjected with following table. C01 intraperitoneally and the amounts of triglycerides TABLE 2 and phospholipids in the liver was determined at 6 or 24 hours after CCL; administration. The results are shown in Meani the following table. Number ofmice GOT GPT TABLE 0 GFUA p-EPH plus c014 5 0. 25i0.06 0.070.04 Time C014 only 5 4.38:l:1.35 2. 65:1:0-48 alter Phos ho- Normal control 5 0. 11:1:0. 01 0. 03:1:0. 02 01 11 1 admin, No.0! Trl lycerldesp mg./g. wet 1 Unit in mole oxalacetate/min/mi. serum. hrs. mice mg. g. wet liver liver As is clear from the table, the elevation of serum trans- GFUA p- 4- 6 5 48d= 1 35 8:1:2 9 h c 1 0 y 6 5 00:1: 34441.1 amlnase actlvltles was reversed to normal by t e admm 8gP m cchm g lggfig} (d) 25% 0 Y stratum of GFUA Normal control 4 30::7 32.8=l=2.9

(b) Serum transamlnase (rat): Four or SLX female ra (Wistar-Imamichi strain, 195-295 g.), having received cigfg f fi GFUA p-EP-H (l00mg./kg.) orally 1 hour before, were -24 hrs. after vehicle given.

4 Difference significant at P 0.0l.'

injected with CCL, intraperitoneally.

(f) Liver protein synthesis (mouse): Four male mice (ddY-strain, about 20 g.), having received GFUA p-E'PH i orally .30 minutes before, were injected with CC]; (1.5 omits GOT GPT mL/kg.) intraperitoneally. p GFU A p-EPH plus After 1 or 2 hours, phenylalanine-TLC Was injected 5 533311 251159 4" aoisflz: 23:57:: intraperitoneally to the micein a dose of l i Ci/ g. and 1;-5i- 475:6 H Phenylalanine-U-C incorporated into 'trichloroacetic fiffifggggg f TC 265:5} (I) acid-insoluble protein of the liver was determined at 6111' l I 4 25:10

minutes after the injection of phenylalanine U-C The 10 1 o5 results are shown in the-following table.

NOTE.M98I1:I:S.D. Karmen Units in 11 times diluted serum.

TABLE 7 Incorporation per Protein per Incorporation per Protein per mg. protein g. liver mg. protein g. liver 1 hour after 0014, GE UA p-EPH, 100 mgJkg, p.o.:

Normal eontro .4. 10019 21. 85:24 100=I=6 54.7=l=1.9 C014 only Y 66. 3=l=5.6 (I) 21. are 67. 75:15. 5 53.9;h2.4 GFUA p-EPH plus COl4-. 125:i=23 24. 45:1.4 116=|=6 52. :1. 0 GFUA p-EPH only 104:i:19 28. 4i2. 1 117=t7 60. 3:l:2. 7 2 hours after 0014, GFUA p-EPH; 50 mg./kg., p.o.:

Normal control -r. l00=l=22 Z6.5;l=3.0 100:1:23 75. 4:231 0014 only 46. 0:l:13.2 21. 6111.9 65.5:l=12.0 55.7;b4. 6 GFUA p- EPH plus C014 75. 05:15. 7 29. 1:1:2. 9 97. 8;i=9. 4 63. 8:1:4. 0 GFUA p-EPH only t11?Orin-Incorporation oi the radloaetivlty was expressed relative to the values for respective normal control. Protein, mgJg. we ver.

As is clear from the table, the decrease of liver protein synthesis, one of the earliest disturbance, was reversed 35 (4) Liver injuries due to long-term treatment with or attenuated by administration of GFUA p-EPH. CCl .-The results of an eight-week study, using female 4 (2) Thiacetamide-induced acute liver injuries-Five rats treated with 1.5 m-L/kg. of CCL; subcutaneously twice male mice (ddY-strain, 20-30 g.), having received GFUA a week, show marked reductions of liver fibrosis as well p-EPH (35.6 mg./ kg.) orally 30 minutes before, were as vacuolization and/or fatty changes of hepatic cells due inpected with thioacetamide (200 mL/kg.) intrapen'to- 40 to daily coadministration of 40 mg./kg. of GFUA p-EPH neally. (Table 10).

TABLE 10 Influence of GI UA p-EPH ln Och-Intoxicated Rat Liver N 3 weeks 6 weeks 8 weeks CCli %15iml./100 g. 2X/w.:

Vacuolization fatty change ++I-++ i-I- iii i i i Col ax? pEPH4mg./l00 g. daily: 1 d: i l H :b

Vaeuolization fatty change :i: =1: :1: :1: i+

After 24 hours, serum transarninase (glutamic-oxalacetic (5) Liver injuries due to long-term chloride detransaminase and glutamic-pyruvic transaminase) activificiency.Male and female rats were fed with a ch01ineties were determined. The results are shown in the foldeficient diet for 5 months. A group of rats were given lowing table. V, v additionally GFUA p-EPH in a dose of 8 or 40 mg./kg.

TABLE 8 for the last 3 months. The histopathological results listed in Table 11 indicate that GFUA p-EPH is also effective w in this form of nutrient-deficient liver injuries. l GOT GPT Thioacetamide my 1.16- l=0.60 0.66:l:0.28 TABLE GFUA p-EPH plus thioacetamide 0.2051006 0.09=l=0.01 Influence of GFUA p-EPH on Choline Deficient Rat Liver Normal control-.. 0.11i0.0l 0.03=l=0.02

. Vacuolization and/or N0'rE.-MeauiS.D. ot5animals. P rt 1 fib i attyr ohtlrlnge oi 08 I055 6310095 As 1s clear from the table, the elevation of serum transp aminase activities was reversed to normal. I-Iistopathoi logical examinations revealed no signs of abnormalities'in Choline deficient (a 1 '2 3- l a o' 7 4 4 th li r e I V 5 ,months) rats (17) y} (9) (15) t v k Raton chohue deficlent (3) Ethiomne-mduced acute l1ver 1n ur1es.-Four or six diet (s i o nhs) and daily;

cm a nistration o v i female rats (Wrstar Imarmchr strain, 195 295 g.), starved GFUA for his 1 0 o v 3 1 1 0 for 16 hours before the experiment, were used. monthsii I (1) I Four or six rats,.having received .GFUA p-EPH (100 ring/kg.) orallyl hour before, were injected with ethiog nine intraperitoneallyu l V EXPERIMENT 3 After 24 hours, serum transaminase (glutamic-oxali i i i acetic transaminaseand aglutamic-pyruvic transamiuase) g i 1 activities were determined. The results are shown in the Acute toxicity was examined by administering ethyl-D- following table. glucuronoside-p-ethoxyphenylhydrazide and ethyl-D-glu- Nora-Number oi rats in parentheses.

curonoside-p-chlorophenylhydrazide orally to male mice ddY-strain,. 25-30..g.)...The results are shown in thefo1- lowing. I

V V V M (a) ltiortalltyaiter liours I V Dose, g./kg.

Ethyl-D-glucuronoslde-p-ethoxy- Y /3 0/3 0/3 0/2 on of? 7/7 pheuylhydrazlde Ethyl-D-glucuronoside-p-chlorophenylhydrazlde 0/8 0/3 0/3 0/2 0/1 2/4 6/6 (b) Toxic symptoms Ethyl-D-glucuronoslde-pethoxy-phenylhydrazide:

10 g.lkg Cyan0sis(7/ 7), convulslon(4fl, tremor phenylhydrazide (10 gJkg.) 4 1 0 1 0 0 Subacute toxicity An equal number of male and female rats (2 x 4 x 10) of Wistar-Imamichi strain weighing between 150 and 190 g. received 125, 250 or 500 mg./kg. of GFUA p-EPH or physiologic saline intraperitoneally daily for 30 days. Body weights recorded on the 8, 20 and 30th day indicated no difference between the control and the treated groups. Hematological studies performed on the 15th and 30th day revealed to abnormalities in terms of number of RBC, hemoglobin, number and pictures of WBC. Serum activities of alkaine phosphatase, GOT and GPT, and serum concentrations of BUN and total protein were determined on the 30th day. Approximately two to three-fold increases of GOT activities were noted with the highest dose groups, as listed below in the order of control, 125, 250 and 500 mg./kg. (this pattern will also be followed in the subsequent description): male, 57.4259 (SD. of 5 animals), 93.7:23.1, 97.3:150 and 163:57; female, 600:8.0, 71.71173, 65.8:98

and 118:23 Karmen units. No abnormalities-were found with the other four serum values. The Weights of organs (heart, liver, kidneys, testes (or ovaries), adrenals, pituitary, brain) on the 30th day were found in the normal range except for those of the spleen which increased approximately three-fold at the highest dose level in the males: 655:84 (SD. of 5 animals), 9101102, 1029250 and 1638:138, while an increase of only 40% was seen in the females: 5561-68, 6781-87, 879:53 and 783x101 mg. Only minor degrees of histopathological changes were detected at the highest dose level in the liver (vacuolization, 1/10; congestion, 1/10), kidneys (vacuolization or peeling of renal tubule epithelia, '5/10), spleen (congestion, 5/ increase of reticular cells, 4/ 10; hemosiderosis, 2/10; follicular cell degeneration, 2/10). No histopathological abnormalities were found in the lungs, heart, digestive tract, brain, pituitary and adrenals.

EXAMPLE 1 3.8 g. of methyl-p-D-glucofuranosiduronolactone was dissolved in 5 ml. of butanol and to the solution was added 4.3 g. of phenylhydrazine followed by .heating on a water bath for two hours. The reaction mixture was .then concentrated under reduced pressure and ether 'was added to the residue. The precipitated crystalline methylfi-D-glucurondside plienylhydrazide was recrystallized from ethyl acetate to give 4.2 g. of the product having a melting point of 130 13? C.

Analysis.Calcd forC H 0 N (percent): C, 52.34; H, 6.08; N, 9.39. Found (percent): C, 52.50; H, 6.13; N, 9.27.

8 EXAMPLE 2 4.2 g. methyl-fl-D-glucofuranosiduronolactone was suspended in 10 ml. .of butanol and to the suspension was added 4.9 g. of o-tolylhydrazine followed by heating on a boiling water bath for two hours while stirring. The reactionmixture was then concentrated under reduced pressure and dichloromethane was added to the residue to give crystalline methyl-fl-D-glucuronoside-o-tolylhydrazide. Recrystallization from isopropyl alcohol yielded 5.1 g. of the product having a melting point of 130-l32 C.

Analysis.-Calcd for C H O N (percent): C, 53.84; H, 6.45; N, 8.97. Found (percent): C, 53.61; H, 6.38; N, 8.92.

EXAMPLE 3 5.4 g. of methyl-p-D-glucofuranosiduronolactone was added to 6.3 g. of m-tolylhydrazine and the mixture was heated on a boiling water bath for one-half hour while stirring to precipitate crystalline methyl-B-D-glucuronoside-m-tolylhydrazide. The resulting product was then washed with chloroform and recrystallized from ethanolether to give 6.8 g. of the product having a melting point Analysis;--Calcd for C H O N (percent): C, 53.84; H, 6.45; N, 8.97. Found (percent): C, 53.44; H, 6.42; N, 8.81.

EXAMPLE 4 3.8 g. of p-tolylhydrazine hydrochloride was suspended in 20 ml. of ethanol and to the suspension was added a solution of 0.56 g. of sodium metal in 20 ml. of ethanol. The precipitated sodium chloride was filtered off, and 3 g. of methyl- -D-gluoofuranosiduronolactone was added to the filtrate followed by heat-refluxing for two hours. The reaction mixture was then concentrated under reduced pressure, and the residue was dissolved in hot chloroform. After allowing to cool, the precipitated crystalline methylfl-D-glucuronoside-p-tolylhydrazide was recrystallized from butanol-isopropyl ether to give 3.2 g. of the product having a melting point of l40l42 C.

Analysis.-Calcd for C H O N (percent): C, 53.84; H, 6.45; N, 8.97. Found (percent): C, 54.00; H, 6.43; N, 9.11.

X M E 5 5 g. of 3,4-dimethylphenylhydrazine was dissolved in 10 ml. of butanol and to the solution was added 4 g. of methyl-fl-D-glucofuranosiduronolactone followed by stirring on a boiling water bath for two hours. The precipi- 'tated crystalline methyl-B-D-glucuronoside-3,4-dimethylphenylhydrazide was recrystallized from isopropyl alcohol to give 4.7 g. of the product having a melting point of 163-165" C.

Analysis.Calcd for C H O N (percent): C, 55.20; H, 6.80; N, 8.58. Found (percent): C, 55.20; H, 6.74; N, 8.66.

' EXAMPLE 6 4 g. of p-ethylphenylhydrazine was dissolved in 10 ml. of butanol and to the solution was added 3.8 g. of methylfi-D-glucofranosiduronolactone followed by stirring on a boiling water bath for two hours. Dichloromethane was then' added to the reaction mixture to give crystalline methyl fi-D-glucuronoside-p-ethyl-phenylhydrazide. Recrystallization from butanol yielded 5.1 g. of the product having a meltingpoint of 147-150 C.

' Analysis.Calcd for C H O N (percent): C, 55.20; H, 6.80; N, 8.58. Found (percent): C, 55.60; H, 6.94; N, 8.71.

' EXAMPLE 7 450 mg. of o-methoxyphenylhydrazine was dissolved in 1.5 ml. of butanol and to the solution was added 400 mg. of methyl-a-D-glucofuranosiduronolactone followed by 'stirring on a boiling water bath for 1.5 hours. The reaction mixture was concentrated under reduced pressure and ether was added to the residue to give crystalline methyl-ot-D-glucuronoside e- 'methoxyphenylhydrazide. Recrystallization from ethanol-ether yielded 470 mg. of the product having melting point 143144.5 C. and [u] +98 (c., 0.5 in H O).

Analysis.Calcd for C gH O N (percent): C, 51.21; H, 6.14; N, 8.53. Found (percent): C, 51.10; H, 5.95; N, 8.54.

EXAMPLE 8 5 g. of o-methoxyphenylhydrazine was dissolved in 7 ml. of butanol and to the solution was added 5 g. of methyl-B-D-glucofuranosiduronolactone followed by stirring on a boiling water bath for 1.5 hours. The reaction mixture was then concentrated under reduced pressure, and ether was added to the residue to give crystalline methyl-B-D-glucuronoside o methoxyphenylhydrazide. Recrystallization from ethanol yielded 6.7 g. of the product having melting point 148-150 C. and [a] l6 (c., 0.5 in H O).

Analysis.Calcd for C ll- 0 M, (percent): C, 51.21; H, 6.14; N, 8.53. Found (percent): C, 51.54; H, 6.16; N,

EXAMPLE 9 8 g. of p-bromophenylhydrazine was dissolved in 15 ml. of ethanol and to the solution was added 6 g. of methylp-D-glucofuranosiduronolactone followed by heat-refluxing on a water bath for four hours. The reaction mixture was concentrated under reduced pressure, and chloroform wasadded to the residue to give crystalline methyl-fl-D- glucuronoside-p-bromophenylhydrazide. Recrystallization from ethanol-chloroform yielded 7.2 g. of the product having a melting point of l69-171 C.

Ana'lyts'is cjalcd for C OgI-I N Br (percent): C, 41.40; H, 4.54; N, 7.43. Found (percent): C, 41.17; H, 4.44; N, 7.13.

EXAMPLE- 10 5.3 g. of 3,4-dichlorophenylhydrazine was dissolved in 10 ml. of butanoland to the solutio'nwas added 3.8 g. of methyl-fl-D-glucofuranosiduronolactone followed by stirring on a boiling water bath for two hours. The reaction mixture was concentrated under reduced pressure, and chloroform was added to the residue. The precipitated crystals were recrystallized from ethyl acetate to give 5.5 g. of crystalline methyl-B-D-glucuronoside-B,4-dichlorophenylhydrazide containing /2 molecule of crystal water: melting point, 164-167 C.

Analysis.Calcd fOl' C H O N Cl 2H O (percent): C, 41.51; H, 4.55; N, 7.45; CI, 18.85. Found (percent): C, 41.49; H, 4.45; N, 7.44; Cl, 18.78.

EXAMPLE 11 20 g. of D-glucuronolactone was suspended in 400 ml. of ethanol and to the solution was added 10 g. of an ionexchange resin (Amberlite 1R-120 B, H-type, available from Rohm & Haas Company) followed by heat-refluxing for six hours while stirring. The mixture was then filtered and, after addition of 22g. of phenylhydrazine to the filtrate, the mixture was refluxed for two hours. The reaction mixture was concentrated under reduced pressure, and the residue was recrystallized from chloroform to give 'ethyLD-glucuro glucuronoside-phenylhydrazine (a mixture, of mand fl-forrns). Recrystallization from ethanol yielded 27.2 g. of the product having a melting point of 137139 C.

Anfalysis.Ca1cd for C H O N (percent): C, 53.84; H, 6.45; N, 8.97. Found (percent): C, 54.01; H, 6.68; N, 9.17. I

. EXAMPLE 12 10 g. of ethyl-D-glucofuranosiduronolactone (a mixture of uand B-forms) was dissolvedin 40 ml. of butanol and to the resulting solution was added 9 g. of p-tolylhydrazine followed by heat-refluxing for two hours. The react-ion mixture was then concentrated under reduced pressure, and the residue was washed with ether-petroleum ether and dissolved in hot dioxane. The solution was then allowed to cool to give 5.7 g. of crystalline ethyl-D- glucuronoside-p-tolylhydrazide containing /2 mole of dioxane (a mixture of aand ,B-forms), melting point 135 137 C.

Analysis.-Calcd fol. C15H22O5N2'1/2 C HgOz (P6P cent): C, 55.12; H, 7.08; N, 7.56. Found (percent): C, 55.17; H, 7.37; N, 7.56.

EXAMPLE 13 9.5 g. of ethyl-D-glucofuranosiduronolactone was dissolved in 20 ml. of butanol and to the solution was added 9.5 g. of p-ethoxyphenylhydrazine followed by heating on a water bath for two hours. The reaction mixture was concentrated under reduced pressure, and ether was added to the residue to give crude crystalline ethyl-D-glucuronoside-p-ethoxy-phenylhydrazide. Recrystallization from butanol yielded 1.6 g. of crystals having melting point 173174 C. and {ed- (c., 0.5 in H O). The mother liquor from the recrystallization was concentrated under reduced pressure and the crystals which were obtained by adding ether-petroleum ether to the residue were recrystallized from ethyl acetate and then from butanolisopropyl ether to give a second crop of 9.5 g. crystals having melting point 131132 C. and Doe-1 -16 (0., 0.5 in H O). The thus obtained first and second crops were considered to be a-form and ,B-form, respectively, and the infrared absorption spectra (KBr) of the products are shown in FIG. 1 and FIG. 2, respectively.

Analysis.-Calcd for C H O N (percent): C, 53.92; H, 6.79; N, 7.86. Found (percent): C, 54.21; H, 6.71; N, 8.04 (for ot-form). Found (percent): C, 54.16; H, 6.93; N, 7.98 (for fi-form).

EXAMPLE 14 12 g. of ethyl-D-glucofuranosiduronolactone was dissolved in 20 ml. of butanol, and to the solution was added 12.5 g. of p-chlorophenylhydrazine followed by heatrefiuxing for two hours. The reaction mixture was concentrated under reduced pressure, and the residue was washed with ether-petroleum ether and then crystallized from chloroform to give 16 g. of crude crystals of ethyl-D- glucuronoside-p-chlorophenylhydrazide. The thus obtained product was recrystallized first from ethyl acetate and then from ethanol yielded crystals having a melting point of 182-183 C. and [a] +l02 (c., 0.5 in methanol) which is considered to be a-form. Also, the mother liquor from the ethyl acetate recrystallization was cooled to give crystals having a melting point of 135.5-137" C. and [@10 i- 25 (c., 0.5 in methanol) which is considered to be B-form.

Analysis.Calcd for C H O N CI (percent): C, 48.50; H, 5.52; N, 8.05. Found (percent): C, 48.68; H, 5.56; N, 8.02 (for a-form). Found (percent): C, 48.67; H, 5.57; N, 8.02 (for B-fortn).

EXAMPLE 15 10 g. of ethyl-D-glucofuranosiduronolactone (a mixture of aand [i-forms) was dissolved in 20 ml. of chloroform and to the solution was added 12 g. of p-bromophenylhydrazine followed by heat-refluxing on a water bath for six hours. Chloroform was then added to the reaction mixture to give crystalline ethyl-D-glucuronoside-p-bromophenyl hydrazide (a mixture of aand B-forms). Recrystallization from ethyl acetate-n-hexane yielded 11.6 g. of the product having a melting point of 134-137" C.

Analysis.-Calcd for C H O N Br (percent): C, 42.98; H, 4.90; N, 7.16. Found (percent): C, 42.81; H, 4.81; N, 6.89.

EXAMPLE 16 3.1 g. of D-glucuronolactone was suspended in 40 ml. of n-butanol and to the suspension was added 1.5 g. of an ion-exchange resin (Amberlite IR- B, H-type, available from Rohrn & Haas Company) followed by refluxing for six hours while stirring. The resin was filtered off from the reaction mixture and, after concentrating the filtrate to about 10 ml., 4.5 g. of phenylhydrazine was added to the filtrate followed by heating on a boiling water bath for two hours. The reaction mixture was then concentrated under reduced pressure, and the residue was crystallized from ether-petroluem ether. Recrystallization of the thus obtained crystalline product from butanol-isopropyl ether yielded 4.2 g. of butyl-D-glucuronoside-phenylhydrazide, melting point 137138 C.

Analysis.Calcd for C H O N (percent): C, 56.46; H, 7.11; N, 8.23. Found (percent): C, 56.78; H, 7.32; N, 8.25.

EXAMPLE 17 To a solution of 4 g. of butyl-D-glucofuranosiduronolactone in 10 ml. of butanol was added 5 g. of p-tolylhydrazine followed by heating on a water bath for two hours. The reaction mixture was then concentrated under reduced pressure, and the residue was crystallized from etherpetroleum ether. Recrystallization from butanol-isopropyl ether yielded 4.7 g. of butyl-D-glucuronoside-p-tolylhydrazide having a melting point of 136-138 C.

Analysis.--Calcd for C H O N (percent): C, 57.61; H, 7.40; N, 7.91. Found (percent): C, 57.39; H, 7.38; N, 7.91.

EXAMPLE 18 To a solution of 4 g. of butyl-D-glucofuranosiduronolactone in ml. of butanol was added 6 g. of p-chlorophenylhydrazine followed by heating on a water bath for two hours. The reaction mixture was then concentrated under reduced pressure, and a mixture of ether-petroleum ether was added to the residue to give butyl-D-glucuronoside-p-chlorophenylhydrazide. Recrystallization from butanol-isopropyl ether yielded 5.2 g. of the product having a melting point of 164-165 C.

Analysis.-Cald for C H O N Cl (percent) C, 51.27; H, 6.18; N, 7.48. Found (percent): C, 51.49; H, 6.28; N, 7.66.

EXAMPLE 19 5 g. of m-nitrophenylhydrazine was dissolved to 10 ml. of butanol and to the solution was added 3.5 g. of methylfl-D-glucofuranosiduronolactone followed by heating on a boiling water bath for four hours while stirring. The reaction mixture was cooled to give crystalline methyl-p-D- glucuronoside m-nitrophenylhydrazide. Recrystallization from isopropyl alcohol yielded 3.7 g. of the product having a melting point of 153-155 C.

Analysis.-Calcd for C H O' N (percent): C, 45.48; H, 4.99; N, 12.24. Found (percent): C, 45.22; H, 4.88; N, 12.04.

What is claimed is:

1. A glucuronic acid derivative of the formula wherein R is a lower alkyl group, and R is a phenyl group which may contain one or more substituents selected from the group consisting of a lower alkyl group, a lower alkoxy group, a nitro group, a chlorine atom and a bromine atom.

2. A glucuronic acid derivative according to claim 1 wherein the phenyl group of R contains one or two of said substituents.

3. A glucuronic acid derivative according to claim 1 wherein the phenyl group of R contains one of said substituents at one or two positions thereon.

4. Methyl-D-glucuronoside phenylhydrazide in accordance with claim 1.

5. Methyl-D-glucuronoside-o-tolylhydrazide in accordance with claim 1.

6. Methyl-D-glucuronoside-m-tolylhydrazide in accordance with claim 1.

7. Methyl-D-glucuronoside-p-tolylhydrazide in accordance with claim 1.

8. Methyl D-glucuronoside-3,4-dimethylphenylhydrazide in accordance with claim 1.

9. Methyl D-glucuronoside-p-ethylphenylhydrazide in accordance with claim 1.

10. Methyl-D-glucuronoside o methoxyphenylhydrazide in accordance with 1.

11. Methyl D-glucuronoside-p-bromophenylhydrazide in accordance with claim 1.

12. Methyl D-glucuronoside-3,4-dichlorophenylhydrazide in accordance with claim 1.

13. Ethyl-D-glucuronoside-phenylhydrazide in accordance with claim 1.

14. Ethyl-D-glucuronoside-p-tolylhydrazide in accordance with claim 1.

15. Ethyl-D-glucuronoside-p-ethoxyphenylhydrazide in accordance with claim 1.

16. Ethyl-D-glucuronoside-p-chlorophenylhydrazide in accordance with claim 1.

17. Ethyl D glucuronoside-p-bromophenylhydrazide in accordance with claim 1.

18. Butyl-D-glucuronoside-phenylhydrazide in accordance with claim 1.

19. Butyl-D-glucuronoside-p-tolylhydrazide in accordance with claim 1.

20. Butyl-D-glucuronoside-p-chlorophenylhydrazide in accordance with claim 1.

21. Methyl-D-glucuronoside-m-nitrophenylhydrazide in accordance with claim 1.

22. A process for preparing a glucuronic acid derivative according to claim 1 consisting of reacting an alkyl- D-glucofuranosiduronolactone of the formula 1!; wherein R is as defined in claim 1, with a phenylhydrazine compound of the formula H N-NHR wherein R is as defined in claim 1.

23. A process for preparing a glucuronic acid derivative according to claim 2 consisting of reacting an alkyl- D-glucofuranosiduronolactone of the formula wherein R is as defined in claim 2, with a pheny1hydra zine compound of the formula H wherein R is as defined in claim 3, with a phenylhydrazine compound of the formula H N-NHR;,

wherein R is as defined in claim 3.

25. A process according to claim 22, wherein the reaction is carried out in the presence of an organic solvent.

26. A process according to claim 25, wherein said organic solvent is selected from the group consisting of a1- cohol and chloroform.

27. A process according to claim 22, wherein said phenylhydrazine is used in an equimolar amount or in excess with respect to the alkyl-D-glucofuranosiduronolactone reactant.

28. A process according to claim 22, wherein said re- 10 14 References Cited UNITED STATES PATENTS 3,284,439 11/1966 Argoudelis 260-210 R OTHER REFERENCES Noller: Chemistry of Organic Compounds, 3rd ed., 1965, W. B. Saunders Co., Philadelphia, Pa.

JOHNNIE R. BROWN, Primary Examiner US. Cl. X.R. 424-180 

